Fine metals (including, for example, fine metals having surface structures of the scale of nanometers and fine metal particles of the size of nanometers) exhibit a characteristic optical response (optical absorption), called “localized (surface) plasmon resonance absorption,” in a specific wavelength range, according to their shape and size. Examples of metals exhibiting localized plasmon resonance absorption include precious metals such as gold, silver and platinum, and it is important that the plasmon resonance absorption wavelength changes according to the size and shape even for the same kind of metal. Utilizing this feature, applications of fine metals and fine metal particles such as described above to various kinds of optical devices (for example, optical filters) are anticipated.
Further, there are important applications of localized plasmon resonance absorption. The intensity of optical response (including, for example, fluorescence and light scattering (Raman scattering)) of molecules adsorbed to the metal exhibiting plasmon resonance is enhanced significantly by interactions between the molecules and surface plasmon. In other words, a metal structure, which is prepared by forming a metal that exhibits plasmon resonance on a substrate, functions as a high sensitivity sensing device for molecules, and application, research and development in this field are studied actively.
Raman scattering light is usually a weak optical response and should be enhanced efficiently to be used as a response signal of a sensor. Therefore, it has been difficult to use Raman scattering light as a response signal of a sensor even by enhancing with plasmon.
On the other hand, fluorescence is an optical response that can be obtained when the sensing-target object is fluorescent molecules or fluorescent-labeled molecules, and usually provides a stronger signal than scattering light such as in Raman scattering. Accordingly, fluorescence is suitable to be used as a response signal of a sensor by enhancement by plasmon resonance. It is therefore possible to detect fluorescent molecule or fluorescent-labeled molecule adsorbed to a metal structure exhibiting plasmon resonance absorption, with the help of fluorescence as a response signal. However, fluorescence (exciting light) from such fluorescent molecule adsorbed to a metal structure is quenched by energy transfer from the fluorescent molecule to fine metal (fine metal particle) adhered with the fluorescent molecule or the like even by being enhancing with plasmon, and is often difficult to use as a response signal.
Further, in fluorescent sensing, there are cases where an impurity (interfering substance) in the target system is excited by the excitation light for exciting the sensing-target substance. When the excited impurity is a fluorescent substance, the fluorescence from the interfering substance becomes an intense background signal (background light) and significantly degrades the sensing sensitivity and sensing precision for the sensing-target substance. Important examples of application of fluorescent sensing include biological analysis such as immunoassay analysis and gene (DNA) analysis, and, in such biological analysis, particularly, the fluorescence from the interfering substance (including serum and enzyme) becomes background light, resulting in a serious problem of causing deterioration of analysis precision.
To avoid the above problem of background light, a sensing method using two-photon excitation is proposed. Two-photon excitation refers to the phenomenon where an electronic excited state (i.e. fluorescent state) is induced by irradiating a light to sample, wherein the light has a wavelength twice the light wavelength (i.e. energy per photon is half) which can induce electronic transition of a fluorescent labeling reagent or fluorescent substance and making one molecule absorb two photons. By irradiating this two-photon excitation to a surface plasmon enhancing sensor, only a portion where the electric field of the excitation light is strongly localized by resonance plasmon is selectively excited. Therefore, it is possible to significantly reduce the influence of background light caused by interfering substance such as described above, and significantly improve sensing sensitivity and sensing precision.
As a technique to detects optical response using two-photon excitation, for example, there is the technique described in Patent Document 1.
Patent Document 1 discloses a fluorescent immunoassay analysis technique for supplying a sample solution containing an antigen to a thin metal film with an antibody fixed thereto so as to bond the antigen and antibody, supplying a fluorescence labeling reagent containing a fluorescent labeling antibody so as to bond the fluorescent labeling antibody to the antibody bonded to the antigen, irradiating light with a wavelength of an integral multiple of the wavelength of light that the fluorescent labeling antibody ordinarily absorbs to a glass prism having the thin metal film, inducing two-photon excitation or multiphoton excitation, and analyzing the spectrum of emitted fluorescence.
Patent Document 1: Japanese Patent Application Laid-Open No. 2001-021565